Every bloody protocol suggests adding in DTT when doing in vitro RNA transcription. Why? The rationale seems to be that the cytoplasm traditionally has a reducing environment but as the only protein we care about is the T7 polymerase, why is this necessary.

You're right. I just looked up a couple of protocols (an example from Promega ) and it has a final concentration of 10 mM DTT. They don't explain why, however it must be important for it to be included.
–
leonardoMar 27 '12 at 22:56

1 Answer
1

Bacteriophage T7-induced DNA polymerase is composed of a 1: 1
complex of phage-induced gene 5 protein and Escherichia coli
thioredoxin. Preparation of active subunits in the absence of
sulfhydryl reagents indicates the reduced form of thioredoxin is
sufficient for formation of the active holoenzyme. The oxidized
form of thioredoxin, thioredoxin modified at one active site
sulfhydryl by iodoacetate or methyl iodide, or thioredoxin modified
at both active site sulfhydryls by N-ethylmaleimide, are all
inactive, being defective in complex formation with gene 5
protein.

General Requirements-The general requirements for T7 RNA
synthesis directed by T7 DNA polymerase are shown in Table
I. As expected for a template directed polymerase, RNA
synthesis shows an absolute requirement for DNA, the 4
ribonucleoside triphosphates and Mg++.

(no surprises there ;)

The activity of the
enzyme is reduced significantly if a sulfhydryl reducing
agent such as b-mercapto-ethanol is omitted from the
reaction. The addition of 10^-5 M p-hydroxymercuribenzoate to
the assay system in the absence of b-mercaptoethanol
abolished all activity, indicating that the enzyme contains a
sulfhydryl group necessary for activity.

However, if you see the table I, the remaining activity after removing bme is still 74%

How strange. I'm looking at the crystal structure (1QLN) and there aren't a lot of cysteines near the active site. Will have to take a look at those papers.
–
bobthejoeMar 28 '12 at 0:39

@jp89 Thanks for the copy of the paper. I'm not sure though if this is allowed here, since it is breaking copyrights. Anyhow, for now I believe it is still fair use. Regarding to the question, I'd follow citations 59-62 from there (last paragraph on page 2/first on page 3). Cheers.
–
AleadamMar 28 '12 at 1:10

Bacteriophage polymerase is active within the cell, which has a reducing environment. Most protocols of intracell proteins include DTT or glutathione for this reason. Disulfides are mostly for secreted proteins.
–
shigetaMar 28 '12 at 13:16

@Aleadam Yeah, that is the sad case. I honestly don't understand the concept of copyrighting human knowledge.
–
jp89Mar 28 '12 at 15:36